Hematopoietic stem cells (HSCs) undergo dramatic expansion in the fetal liver. The long term goal of this proposal is to characterize the stromal cells for HSC expansion in fetal liver and identify the growth factors they secrete for expanding HSC numbers. This study will not only answer important basic biological questions, but it can lead to major improvements in the clinical use of HSCs in bone marrow transplantation for treatment of leukemia and other cancers. HSCs are also a promising cell target for developing gene therapies for treating a broad variety of inherited immunodeficiency syndromes and inborn errors of metabolism. Previous studies and our preliminary data indicate that certain subpopulations of fetal liver E15.5 CD3+ and B220+ cells express high levels of four growth factors: IGF2, AngptI3, SCF and TPO. When added together, these four growth factors are able to significantly expand both adult bone marrow and fetal liver HSCs ex vivo. The cells that express all of these four HSC expansion factors are potential HSC stromal cells since SCF is a transmembrane protein and is proposed to mediate one interaction between HSCs and their stromal cells by binding to its receptor c-Kit, which is expressed by all HSCs. In this proposal, FL B220+ and CD3+ cells will be further fractionated by FACS sorting and mRNAs for many growth factors quantitated by qPCR to determine their ability to produce these four key HSC expansion factors and to discover additional HSC expansion factors they secrete. Competitive repopulating experiment and limiting dilution assay will be used to determine that ability of characterized B220* and CDS* subpopulations to support ex vivo HSC expansion in medium without adding any growth factor. (Specific Aim 1) Parallel studies will also be conducted to determine whether similar cells exist in adult bone marrow and placenta and, if so, whether these cells also express the key HSC expansion factors and can support HSC self- renewal ex vivo. Bone marrow HSCs are normally quiescent;therefore they will also be studied under stressed conditions where they expand in numbers. (Specific Aim2) Finally immunofluorescence staining experiments will be carried out to determine whether characterized CDS* and B220+ subpopulations are located in close proximity to HSCs in fetal liver and thus are bona fide stromal cells for HSCs. (Specific Aim 3) Relevance: The goal of this study is to discover the cells that can cause blood stem cells to dramatically increase in numbers and find out what proteins they use to accomplish this. In the future we can produce the same proteins to generate much more blood stem cells to improve the success rate of bone marrow transplantation for leukemia and cancer as well as develop gene therapies for many inherited diseases.